1. Field of Invention
The present invention relates to immunogenic compositions and to methods for raising antisera to small molecules.
2. Description of Related Art
Antisera are raised by immunising an animal with an immunogen. In general, antisera to a small molecules, such as drugs, cannot be raised using an immunogen comprising the small molecule alone. It has been shown, however, that antisera to drugs may be raised by linking the drug to a large "carrier protein" and immunising animals, for example sheep or rabbits, with the drug-protein conjugate. Reaction Scheme 1 outlines the steps involved in raising antibodies to the drug (F=functional group able to covalently link to protein. The immunogen is prepared by covalently bonding suitably derivatised drug molecules to the carrier protein by means by a bridging group. Antisera are raised in and isolated from animals immunized with the immunogen. ##STR1##
For example, immunogens have been reported for use in raising polyclonal antisera to tricyclic antidepressants (TCA) drugs for use in radioimmunoassays.
Tricyclic antidepressant drugs are a closely related series of compounds with a three ring molecular core. Examples include Desipramine (I) and Nortriptyline (II) (shown below). ##STR2##
Brunswick et al., Life Science (1978) 22: 137-146, have prepared the following immunogen: ##STR3##
This immunogen comprises the TCA drug Nortriptyline bonded, via the tail portion of the molecule, to BSA (Bovine serum albumin), as the carrier protein, by means of a hemisuccinate bridge. The immunogen was used to immunise rabbits.
Read et al., [Post Grad. Med. J. (1977) 53: Suppl. 4: 110-116], have shown that the antisera to TCA drugs may also be raised by immunogens in which the drug is linked to the carrier protein by means of a bridging group bonded to the ring system of the drug: ##STR4##
TCA drugs are widely used for the treatment of major depression. In excessive amounts, however, they cause a number of toxic effects, especially on the heart and, because of the type of patient to whom they are given, are one of the most common drugs taken in suicide attempts. In the USA they account for about 300 drug deaths (accidental and suicide) of a total of 4,000 drug deaths (accidental and suicide) per year.
To be useful in the treatment of a TCA drug overdose the antibodies contained in the antisera must bind the TCA drug thereby reducing the concentration of the free drug in the body. It is calculated, based on an approximate molecular weight of 280 for a TCA drug and an approximate molecular weight of 150,000 for an antibody (whole IgG) that 540 g of antibody would be required to bind 1 g of drug. If, however, just the Fab fragment of the antibody is used in therapy approximately 180 g of Fab would be required to bind 1 g of drug (assuming an approximate molecular weight of 50,000 for a Fab fragment).
The estimation of the amount of TCA drug to be bound by specific Fab in the treatment of an overdose is not simple. The drugs have highly variable volumes of distribution (10-50 l/kg) and, it has been suggested that, neither toxicity nor therapy correlates well with blood concentration [Cantrill et al., J. Emerg. Med. (1983) 1:169-177; and Ware et al., Southern Med. J. (1987) 80:1410-15].
Table I lists the properties of the TCA drugs:
TABLE I ______________________________________ TCA DATA Average Range ______________________________________ Normal Dose 150 mg 25-300 mg Potentially lethal dose 1500 mg 500-10,000 mg Potentially lethal dose kg 20 mg/kg 20-40 mg/kg per Therapeutic circulating 0.15 mg/l 0.05-0.3 mg/l level Toxic blood concentrat- 1.0 mg/l much variation ion Blood concentration (pm 10.0 mg/l 0.47-35 mg/l cases) Tissue concentration 6-26 mg/l (pm cases) Half life Desipramine 18 h Nortriptyline 31 h Volume of distribution 30 l/kg 10-50 l/kg ______________________________________
Using the above figures, it is estimated that between 300 g and 4000 g of specific Fab would be required to neutralise all the drug in an overdose. This may be too large a dose for administration to a patient. If it were found that all the drug in an overdose had to be neutralized to save the patient then this therapy might not be feasible. However, very little of the drug circulates free in the blood where it can cause the most damage (to the heart). It is suggested therefore that a much lower dose of Fab, of the order of 8 g, may be required to save the patient's life.
8 g is a quantity that can reasonably be administered to a patient. Thus, treatment of an overdose requiring binding of the TCA drug in the blood and to reverse the toxic effect on the hear of the patient is a practically feasible proposition.
For production of antisera to TCA drugs to be economically viable however, it is desirable that the titre of the specific antisera raised in, for example, sheep, should exceed 4 g/l.
In addition, it is desirable that the antisera produced bind well to a broad spectrum of TCA drugs.
Known immunogens suffer, however, from a number of drawbacks including first, that animals immunized with these immunogens produce only a low titre of antisera specific to the drugs; and secondly, the antisera produced is of low cross-reactivity, i.e. the antisera exhibits poor reactivity against a series of drug molecules containing small structural variations.
There remains, therefore, a need for immunogenic compositions capable of raising a high titre antisera. There also remains a need for immunogenic compositions capable of raising antisera showing high cross-reactivity with a series of related drugs.